Method for the Analysis of a Blood Sample, and Apparatus and Reagent for Its Implementation

ABSTRACT

Method for the automatic analysis of a blood sample in which: an analysis solution containing the blood sample, a diluent, and at least one compound to lyze the erythrocytes; at least one compound to stabilize the haemoglobin in the form of a chromogenic complex, is formed in a single dilution and analysis tank, the haemoglobin level is measured in this analysis solution by spectrophotometry in the tank after the lysis of the erythrocytes; and an appropriate quantity of this analysis solution is taken from the tank on which a leucocytic differentiation is carried out by an optical elements characterized in that the analysis solution also contains at least one compound to protect the leucocytes, allowing the distinguishing of at least four main leucocyte sub-populations. A haematological analysis apparatus for the implementation of such a method is disclosed.

The present invention relates to a method for the analysis of a bloodsample, as well as an apparatus and reagent for its implementation. Moreparticularly the invention relates to the field of the automaticanalyses of blood samples.

Analysis of a blood sample generally seeks to determine:

-   -   the total number of leucocytes;    -   more specifically, the number of leucocytes by sub-populations        (basophils, eosinophils, neutrophils, monocytes and        lymphocytes);    -   the number of erythrocytes and platelets; and    -   the haemoglobin level.

Several analysis techniques are known, in particular:

assay of the haemoglobin is carried out after lysis of the erythrocytes,i.e. the destruction of the membrane of the cells of erythrocytes, andby measurement by spectrophotometry of the haemoglobin released in themedium; the assay of the haemoglobin also requires the stabilization ofthe haemoglobin in a complexed form (oxyhemoglobin or cyanmethemoglobin)in order to measure the absorbance of a single compound at theappropriate wave length.

total leucocyte count is carried out on the blood sample by resistivitywith specific lysis of the erythrocytes and protection of theleukocytes.

differentiation of the leucocytes and the counting thereof bysub-population is carried out:

-   -   either by resistivity volumetric measurement after specific        lysis of the erythrocytes, protection of the leucocytes and        adjustment of the pH; this however does not allow        differentiation of all the sub-populations in a single analysis;    -   or by optical way, in particular by flow cytometry; after        specific lysis of the erythrocytes and protection of the        leucocytes, by measuring different parameters (in particular        diffraction, fluorescence, absorbance), on a flow of leucocytes        in the axis of the narrow, medium and wide angles and optionally        after addition of a labelling agent (for example chlorazol        black, or a DNA or RNA labelling dye, or a fluorescent dye) and        by measuring at different wave lengths; this technique allows        differentiation of the sub-populations of leucocytes.

the erythrocyte and platelet count is carried out on a diluted samplewithout the addition of specific reagent by resistivity measurement.

Numerous automatic blood cell analyzers exist which use these techniquesin order to obtain a blood sample analysis which is as complete aspossible.

In these automatic apparatuses, two different analysis circuitstraditionally coexist:

a first circuit designed to measure the haemoglobin and/or the totalleucocyte count; and

a second circuit designed to carry out on the blood sample thedifferentiation and/or a leucocyte count by flow cytometry.

Each circuit is characterized by a dilution rate of the blood samplesuited to the measurement means used, the addition of one or morereagents and appropriate means for implementation and measurement.

Thus, for the measurement of haemoglobin and the counting of theleucocytes, the circuit typically comprises a so-called counting tank inwhich the blood sample is diluted, a reagent in particular comprisingthe lysis compound of the erythrocytes, the stabilisation compound ofthe complex formed from the haemoglobin and the leucoprotective compoundis added to it, and the following are measured directly in this cell:haemoglobin by spectrophotometry and the number of leucocytes byresistivity. The dilution rate is chosen so that the analysis solutionis perfectly homogeneous and so that the detection apparatus is notsaturated. This dilution rate is comprised between 1/100^(th) and1/500^(th), generally between 1/160^(th) and 1/180^(th).

For leucocytic differentiation by flow cytometry, the circuit uses atank for dilution of the blood sample to which one or more reagentscontaining an erythrocyte lysis agent, optionally a differentiationagent (for example a DNA or RNA leucocyte fluorescent dye) are added,then a fraction of this solution is taken in order to inject it into aflow-through optical tank of a flow cytometer. The dilution rate usedhere is less than 1/100^(th), allowing an optimal analysis time to beobtained with the cytometers currently available on the market (of thehydrofocus type).

Thus, conventionally, at least two different reagents must usually beused for the two analysis circuits and two different dilutions of theblood sample are carried out in these two analysis circuits.

The main objectives of manufacturers are to simplify the existingautomatic apparatuses by reducing the number of components and,reagents, allowing reduction of the production and maintenance costs andthe size of the automatic apparatuses, without however reducing the timeof a complete blood sample analysis.

The present invention in particular aims to achieve these objectives.

Document WO 2004/003517 for this purpose proposes a method and equipmentin which the two analysis circuits have means in common. The principleis to carry out a first dilution of the blood sample in a singledilution tank and to successively transfer fractions of selected volumesof this dilution to a measuring or counting unit, in order each time tomeasure or count different elements contained in the blood sample. Inorder to carry out a complete analysis, namely counting the erythrocytesand platelets, counting the leucocytes, measurement of the haemoglobinand leucocytic differentiation, the document describes the followingsolution: using a first transfer to count the erythrocytes andplatelets, adding a lysis agent to the dilution tank, then carrying outa second transfer to count the leucocytes, carrying out a third transferof lyzed dilution solution to measure the haemoglobin level, adding aleucocytic differentiation reagent, and carrying out a fourth transferto realize the leucocytic differentiation in the measuring unit.

This principle may allow the use of a single so-called dilution tank,but it does not allow a saving of analysis time because the measurementsor counting are carried out successively after each transfer of afraction of the dilution. Moreover, it requires perfect control of thesuccessive volumes of reagents and diluents transferred to the measuringunit. Moreover it also requires the use of several syringes and lysisreagents.

The objective of the present invention is also to overcome suchdrawbacks.

According to a first object, the present invention relates to a methodfor the automatic analysis of a blood sample and an apparatus forimplementing this method.

In the method according to the invention:

an analysis solution containing said blood sample, a diluent, and:

-   -   at least one compound to lyze the erythrocytes;    -   at least one compound to stabilize the haemoglobin in the form        of a chromogenic complex; is formed in a single dilution and        analysis tank,

the haemoglobin level is measured in this analysis solution byspectrophotometry in said tank after the lysis of the erythrocytes; and

an appropriate quantity of this analysis solution is taken from saidtank on which a leucocytic differentiation is carried out by an opticalmeans.

The method according to the invention is characterized in that theanalysis solution also contains at least one compound to protect theleucocytes, allowing the distinguishing of the five main leucocytesub-populations.

The counting of the leucocytes can be carried out jointly in theanalysis tank and/or with the optical means.

The counting of the erythrocytes and optionally of the platelets can becarried out for example in a previous stage of the method on a samplecarried out in the single dilution and analysis tank.

Thus, the present invention is based on the concept of a single analysissolution used as is for the two types of analyses which were usuallycarried out in two separate circuits, namely on the one hand themeasurement of the haemoglobin and optionally the counting of theleucocytes and, on the other hand, the leucocytic differentiation byoptical means, said analysis solution combining the “reagent” compoundscapable of carrying out at least these analyses by virtue of theirnature and their quantity. The reagent compounds introduced are chosento be chemically compatible with each other and in quantities suited tothe targeted analyses. They can be chosen from the compounds typicallyused in the prior art. It is also possible to use a commercialformulation which is conventionally used to carry out a leucocyticdifferentiation, i.e. containing the compound for lyzing theerythrocytes and the leucoprotective compound, and to add to it thethird reagent compound intended to stabilize the haemoglobin in the formof a chromogenic complex.

Due to this single analysis solution, the present invention inparticular has the following advantages:

-   -   the automatic apparatus can comprise a single tank for        preparation of the analysis solution;    -   the measurement of the haemoglobin can be carried out directly        in this tank, and also the global counting of the leucocytes by        resistivity measurement of the analysis solution;    -   it is possible to use a mono-reagent combining all the “reagent”        compounds required for the measurement of the haemoglobin and        for the leucocytic differentiation by optical means; this in        particular allows simplification of the hydraulic circuits as        will be seen below;    -   a mono-dilution of the blood sample can be carried out directly        in the single dilution and analysis tank, with a dilution rate        determined as a function of the measurement and detection means        used. The mono-reagent can serve as a diluent for carrying out        this mono-dilution. Preferably, a dilution rate will be chosen        comprised between 1/100^(th) and 1/500^(th), corresponding to        the dilution rate required for a measurement of the haemoglobin        level, preferably also a rate of approximately 1/175^(th) (        1/173^(rd) in the embodiment given below).

With the possibility of using a mono-dilution and a mono-reagent, it istherefore possible, thanks to this first aspect of the invention, togreatly simplify the analysis equipment while still providing a completeanalysis of the blood sample.

Means for optical measurement allowing an analysis of the leucocytes(counting and differentiation by sub-populations) at a dilution rategreater than 1/100^(th) are also proposed according to the invention andare defined and described below.

The mono-reagent used in the method according to the invention allowsmeasurement by spectrophotometry of the haemoglobin concentration of ablood sample and a leucocytic differentiation by optical means. It alsoallows the resistive and/or optical counting of the leucocytes.Preferably it is chosen so as to allow the differentiation of at least 5sub-populations. Preferably it is chosen so that it does not containcyanides.

According to the invention, the compound to lyze the erythrocytes ispreferably constituted by at least one cationic surfactant. In apreferential manner which is known per se, it is chosen to form anoxyhemoglobin complex (as it is non-toxic compared to acyanmethemoglobin complex which involves cyanide ions). The cationicsurfactant is therefore also chosen such that it oxidizes the releasedhaemoglobin so as to form only an oxyhemoglobin complex. The quantity ofcationic surfactant is therefore chosen so as to efficiently haemolysethe erythrocytes and oxidize the haemoglobin released. It is preferablychosen from:

the quaternary ammonium salts, preferably alkyltrimethylammonium saltsand still more particularly cetyl-, dodecyl-, tetradecyl- andhexadecyltrimethylammonium bromides and chlorides;

pyridinium salts;

long-chain ethoxylated amines; and

alkyl sulphates (SDS).

The leucoprotective compound according to the invention is a compoundwhich delays or prevents the destruction of the leucocytes. Preferablyit is a non-ionic or amphoteric surfactant preferably chosen from:

ethoxylated alcohols, in particular 2-phenoxyethanol,polyoxyethylenealkylphenylethers, such as the commercial productsIPEGAL990®, TERGITOL NP9®, TRITON® X100 or X114, Plurafac® A38 orBrij35®);

betaines and sulphobetaines of quaternary ammoniums in particularlauramidopropyl betaine (LAB), anddodecyldimethyl-3-ammonio-1-propanesulphonate (DDAPS) ortetradecyldimethyl-3-ammonio-1-propanesulphonate (TDAPS);

tertiary amine oxides, such as N,N-dimethyllaurylamine-N-oxide (LDAO) or3-[(cholamidopropyl)-dimethylamino-]-1-propane sulphonate (CHAPS orCHAPSO);

the glycosidic type compounds and more particularly a triterpenesaponin;

the glucidic type compounds (mannitol, D-glucose, trehalose, dextransulphate).

The compound which stabilizes the haemoglobin in the form of achromogenic complex is preferably chosen from:

mono or polydentate chelates presenting ligand atoms (non-binding pairs:O, N, S, and carboxy COO— groups etc.) in particular:

-   -   ethylene diamine tetraacetic acid (EDTA) or ethylene        glycol-bis-(3-aminoethylether)N-N′-tetraacetic acid (EGTA) and        in particular their sodium or dipotassium salts;    -   potassium oxalate K₂O_(x)O_(x)═C₂Oβ4 ²⁻;    -   hydroxylamine salts (preferably hydrochlorites); and    -   organic acids (in particular formic or acetic).

the aromatic compounds (mono or polydentate chelates) comprising ligandatoms (having non-binding pairs: O, N, S etc.), in particular:

-   -   Tiron®    -   8-hydroxyquinoline and its derivatives;    -   pyridine or bipyridine and their derivatives;    -   1,10-phenanthroline and its derivatives;    -   the phenolic compounds (mono or bis and their derivatives);    -   pyrazole and/or the pyrazolones and their derivatives;    -   imidazole and its derivatives    -   sulphosalicylic acid; and

saponins, tertiary amine oxides, betaines and sulphobetaines ofquaternary ammoniums (such as DDAPS, TDAPS, LAB).

In addition to the three compounds defined according to the invention,it is possible to add to the (mono)-reagent(s):

at least one dye (or mixture) specifically labelling certain leucocytesand more particularly eosinophils (or basophils), in order to allow thedistinguishing of at least the 5 main leucocyte sub-populations, chosenfrom:

-   -   cyanines;    -   Oxazine 750;    -   Wright and Romanowsky reagents;    -   DAPI;    -   Clorazole black E;    -   Toluidine blue;    -   Astra Blue;    -   thiazole orange G. or blue;    -   other fluorescent reagents.

at least one fixing agent allowing stiffening of the membrane of theleucocytes which is preferably an aldehyde and more particularlyglutaraldehyde or formaldehyde;

at least one wetting agent in order to optimize the fluidics and preventthe formation of bubbles which also act as solubilizers of the debris,chosen from:

-   -   alcohols (methanol ethanol or propan-2-ol);    -   glycols (ethylene or propylene glycol);    -   ethoxylated glycols (particularly Triton X100® or Brij35®);    -   glycosidic compounds TWEEN80® or TWEEN20®;        the concentration of the fixing agent and of the solubilizer        being strictly limited, as an excess can prevent the lysis of        the erythrocytes and modify the optical properties of the        leucocytes; and

a buffer system for setting the pH between 5.0 and 10.0 and preferablybetween 6.0 and 8.0 and optimally close to neutrality (7.0±0.4). Thechoice of such a pH aims to respect the native conditions of the cells.Moreover this pH allows a better dissolution of the constituents usedaccording to the invention. Said buffer is constituted by a pair ofsalts (inorganic or organic) adjusted to the above-mentioned pH byhydrochloric acid or soda (4-6N), chosen from:

-   -   sodium or potassium dihydrogen phosphate/hydrogen phsophate        H₂PO₄ ⁻/HPO₄ ²⁻;    -   sodium hydrogen carbonate/carbonate NaHCO₃/Na₂CO₃    -   a citric acid/sodium citrate (III) buffer    -   TRIS-HCl    -   triethanolamine (TEA)    -   imidazole

an acid chosen from:

-   -   the organic acids: phthalic, sulphosalicylic or formic, which        also contribute to the formation and stabilization of the        chromogenic complex of the haemoglobin); and    -   the mineral acids: HCl, H₃PO₄ etc.

a background salt ensuring a conductivity of the order of 10 to 50 ms/cmrequired for the resistivity measurement and an osmolarity of the orderof 120 to 500 mOsm and preferably close to isotonicity (290±5 mOsm),chosen from:

-   -   sodium chloride NaCl;    -   potassium chloride KCl;    -   magnesium chloride MgCl₂;    -   calcium chloride CaCl₂;    -   anhydrous sodium sulphate Na₂SO₄;

this background salt being able to be comprised in the buffer system;

at least one preservative, having antioxidant, and/or antibioticproperties chosen from:

-   -   2-phenoxyethanol;    -   parabens;    -   BHT;    -   isothiazolones (Proclin® 150 or 300);    -   imidazole or urea derivatives;    -   antibiotics;

a natural antibiotic cellular penetration compound (ionophore) whichalso facilitates the penetration of the dye or dyes chosen from:

-   -   ionophore I for NH₄ ⁺ (nonatine);    -   ionophore III for Ca²⁺ (calcimycine);    -   ionophore for Cl⁻;    -   ionophore I for K⁺ (valinomycine).

The constituents according to the invention are summarized in the tablebelow as well as ranges of appropriate concentrations.

Constituent Quantity Cationic surfactant 0.1-50 g/L (lysis agent)Leucoprotective 0.1-20 g/L surfactant Chelate of the 0.0001-10 g/Lhaemoglobin complex Dye 0.01-1 g/L Fixing agent 0.01-2% w/v Wettingagent 0-50% v/v Buffer 0-6 g/L Background salt 1-50 g/L Acid Appropriatequantity to adjust the pH Preservative Appropriate quantity 0.1-3 g/LIonophore Effective quantity 0-200 mg//L Distilled water qsf Qsf 1 L

The present invention also proposes an apparatus for implementing themethod according to the invention which is characterized by:

-   -   an analysis tank which is able to receive said analysis        solution;    -   a means for measuring the level of hemoglobin present in said        analysis solution by spectrophotometry in said tank;    -   a means for sampling said analysis solution;    -   a means for optical measurement on said sample in order to        produce a leucocyte analysis.

According to a second object, the present invention relates to anoptical device for an automatic apparatus for the automatic analysis ofa blood sample, particularly advantageously also for the implementationof the method according to the first object of the invention.

As mentioned above, certain sub-populations of leucocytes can only bedifferentiated by optical measurements, for example a measurement of thediffraction by the cell at one or more angles, or a measurement of theabsorbance of the cell. The optical systems for characterization of ablood cell have a common base in which a light source is locatedemitting a light beam, an optical tank in which the blood cells crossthe light beam, a system for adjustment of the light beam to the flow ofcells and means for measuring the light originating from the opticaltank after interception by the cells. In particular in the case ofleucocyte characterization, the leucocytes move in a flow in the tank.They are illuminated therein by a light beam focussed on the flow, whichis called the sample flow.

Such devices are costly: in particular, the lasers used as lightsources, which are also bulky and generally require a thermaldissipation system; the laser diodes, like the lasers, require costlyalignment systems. The light beams emitted by these sources have atransverse distribution of light which is approximately Gaussian inshape. Thus, the intensity is only approximately constant and maximal ina narrow and central part of the ray. The alignment systems allow thiscentral part to be aligned with the sample flow. Moreover, the width ofthe sample flow must not exceed that of this central part, and thecloser these two widths are, the greater the precision of the alignmentsystem must be. As a result, it is necessary to reduce the width of thesample flow as much as possible.

The sample flow containing the blood cells to be counted and/or to bedifferentiated must be narrower the more the light is focussed. Thus, aflow is used in which the width of the section is less than 50 μm, whichmust cross the light beam which is itself focussed into a narrow beamwith a larger section than that of the sample flow. This requires aparticularly precise and therefore costly system for injection of theflow into the optical tank. In the prior art, such a result is obtainedusing a hydrofocus type system (abbreviation of the English expression“hydrodynamic focusing”). The sample flow is surrounded with a sleevingflow. An injector for the sample flow is immersed in the centre of thesleeving flow. The sample flow thus created is widened or focussed as ittravels from the injector to the zone illuminated by the light beam, sothat it has, at this point, a desired width of approximately 5 to 50 μmin diameter. A single or a double sleeving is sometimes necessary inorder to achieve this objective.

Moreover, as mentioned previously, given the level of precisionrequired, an adjustment system is essential in order for the flow ofcells to be coincident with the light beam. Two approaches are possible:the flow of cells or the light beam can be moved. If it is chosen tomove the flow of blood cells, all of the optical tank unit must bemoved. When this option is adopted, the tank is mounted on a translationtable which ensures a precise and uniform movement along two axes due toits ball bearings. Such a precision mechanical assembly is quite costly.It is also possible to move the light beam in order to make itcoincident with the flow of blood cells. This is generally achievedusing several adjustable prisms. This solution, which combines opticalelements with precision mechanics also involves high costs.

Moreover, when it crosses the light beam, the blood cell deflects thetrajectory of the light rays. The intensity and the angle of thedeflected rays allow information on the cell type to be obtained. Tworanges of angles are generally used: narrow angles less than ten degreeswith respect to the optical axis and wide angles approximatelyperpendicular to the optical axis. In the range of the narrow angles,two items of information are useful: the losses in the axis and thediffraction. Perpendicular to the optical axis, the diffusion and thefluorescence are generally measured. For the two ranges of angles, thelight must therefore be distributed into two different channels. This isgenerally achieved with dichroic mirrors or with interference filters.The optical components are both produced by depositing thin films on aglass substrate. They have good efficiency but a great disparity existsbetween one filter and another and their lifetime is limited. They musttherefore be regularly replaced.

All these generally bulky devices are also fragile and requiremaintenance, which is also very costly. Such devices are thereforerestricted to analytical laboratories which are large enough to be ableto invest in such automatic apparatuses.

The purpose of the invention is to propose a device for leucocytedifferentiation and/or leucocyte counting which is simpler and moreeconomical both to produce and to maintain, allowing the use ofautomatic apparatuses, equipped with the device, by smallerlaboratories, while retaining adequate quality of measurement.

According to the second object of the invention, an optical device forcounting and/or the differentiation of leucocytes in an automatic bloodanalyzer is proposed, characterized in that it comprises a light sourceof the electroluminescent diode type in order to illuminate a bloodsample circulating in the optical tank according to an injection axis,using a source light beam. Such a diode allows a light beam to beobtained which is more homogeneous over the width of its section andtherefore of a larger and more homogeneous reading zone.

Preferably, the diode emits light the wave length of which is less than600 nanometers, and still more preferably less than 500 nanometers. Sucha wavelength allows a better diffraction efficiency, therefore betterprecision for measurements using diffraction.

Moreover, the width of the beam emitted by the optical device, i.e. thesource beam which illuminates the sample flow, is advantageouslycomprised between 50 and 200 microns (μm), close to the injection axis,which allows illumination of a wider sample flow, while allowingadequate precision in the measurements carried out. Yet moreadvantageously, this width is comprised between 90 and 120 microns. Sucha flow width is in particular permitted by the use of electroluminescentdiodes.

Preferably, the source light beam is emitted approximately in thedirection of the tank, approximately transversely to the direction offlow of the sample. A transparent slide designed so that the source beampasses through it between two opposing surfaces, which is rotatablymounted and arranged between the diode and the tank can allow the lightbeam to be moved in a transverse direction, thanks to its doublerefraction when it passes through the slide. The rotation of the slideallows modification of the angle of incidence of the beam on the slide,and thus adjustment of the value of the transverse shift. Preferably,the transparent slide is rotatably mounted about an axis which isapproximately parallel to the movement of the blood sample in the tank.

Beyond the optical tank, means for separation by Fresnel losses areadvantageously used for an incident resulting light beam originatingfrom the source light beam, thus separating said beam into anaxially-resulting beam and at least one beam resulting from lossconstituted by Fresnel losses whilst passing through the separationmeans. The separation means comprise at least one separation surfacewhich is a surface in a transparent separation material, the axial beamhaving passed through the transparent material and the beam originatingfrom the Fresnel losses having been reflected by the separation surface,said surface being slanted in relation to the light beam beyond thetank. A single inexpensive glass slide can serve as separation means.Moreover it has a virtually unlimited and maintenance-free lifetime,unlike dichroic mirrors or interference filters.

The device can also comprise an apparatus for measuring the light of theaxially-resulting beam and at least one other apparatus for measuringthe light of at least one beam originating from the Fresnel losses.These measuring apparatuses can in particular comprise means formeasurement of either the fluorescence, the light losses close to theaxis or the diffraction close to the axis. It can also comprise meansfor measuring the diffraction of the light beam at wide angles by thesample in the tank. By way of example, these wide angles can be anglescomprised between 60° and 150°.

The device can also comprise, in the path of the beam in front of thetank, at least one diaphragm blocking spurious light.

The invention also relates to a hematology apparatus, in particular anautomatic blood analyzer equipped with such a device.

According to a third object, the present invention also relates to aflow-through optical tank for an optical device suitable for thecounting and differentiation of leucocytes, for example a flowcytometer, as well as an analysis apparatus equipped with such a tank.The aim of the invention is to propose a tank which is simpler and moreeconomical both to produce and to maintain, allowing the use ofautomatic apparatuses equipped with this tank by smaller laboratories,while retaining an adequate quality of measurement.

According to the invention, a flow-through tank for an optical devicefor the counting and differentiation of leucocytes in an automatic bloodanalyzer, is characterized in that in an analysis zone of the tank, thesection of the tank has at least one transverse dimension comprisedbetween 1 and 5 millimetres. This section can be approximatelyrectangular and the transverse direction can be measured on one and/orthe other of the sides of the rectangle.

Such a tank can thus be produced, at least partially, from an injectedplastic material. Such a tank is produced in a particularly advantageousmanner compared to the tanks of the prior art, generally formed ofquartz walls assembled by bonding.

The tank can also comprise at least one lens moulded in one piece withthe tank. This at least one lens can comprise a lens envisaged to bearranged laterally in relation to an optical axis. It can comprise ahemispherical lens.

The tank can comprise along an optical axis, a window for theintroduction of a light beam and a window for the beam to exit. At leastone window can be moulded in one piece with the tank and/or be an insertin a transparent material, for example quartz or glass.

The tank can advantageously comprise an injector for a sample flow andmeans for forming a sleeving flow around the injection flow. Theinjector can comprise an outlet orifice the diameter of which iscomprised between 20 microns and 150 microns, allowing a sample flow tobe obtained which is noticeably larger than the flows of the prior art.By contrast to the devices of the prior art, it is not the sleeving flowwhich dictates the width of the sample flow by stretching it, but theshape and the section of the injector outlet. The sleeving flowtherefore does not play an active role, but merely a passive role, inparticular, for example for centering of the sample flow in a wide tank.

According to a first embodiment, this injector can be formed in onepiece in a more or less rigid material. This material can be, forexample, a stainless steel, a ceramic, synthetic ruby or a plasticmaterial or several of these materials.

According to a second embodiment this injector can comprise a rigidstructural tube, for example made of metal, for example made ofstainless steel, and inside the structural tube, a plastic sheathingtube ending in a nozzle formed in one piece with the sheathing tube. Theplastic material of the injector can be a polytetrafluoroethylene, whichallows the sample to circulate more easily in the tube and reduces therisk of fouling up.

The invention also relates to an injector for a tank according to theinvention, which injector is produced according to one of theseembodiments.

The invention also relates to a haematology apparatus, in particular anautomatic blood analyzer, equipped with a tank according to theinvention.

According to a fourth object, the present invention also relates to ahydraulic device for a haematological analysis apparatus, which issimpler and more economical both to produce and to maintain and whichallows the use of automatic apparatuses, equipped with such a device, bysmaller laboratories, while retaining an adequate quality ofmeasurement. The present invention also relates to an analysis methodsuited to such a device.

The present invention thus proposes a hydraulic device for a bloodanalysis apparatus, in particular an automatic apparatus, comprisingmeans for injecting under pressure a sample flow into a flow-throughoptical tank and for creating a liquid sleeving flow around the sampleflow, with a sleeving liquid, characterized in that it comprises meansfor adjusting a flow rate of the sample flow with respect to the flowrate of the sleeving liquid. Such adjustment can make it possible tomaintain homogeneous and approximately non-turbulent flows in the tank.

The injection means can comprise syringes, a hydraulic circuit andsolenoid valves. These means can comprise means for injecting the sampleunder pressure relative to the sleeving flow.

This device can advantageously comprise means for forming a piston forthe sample injected with a displacement liquid. Such a displacementliquid makes it possible to use only a small sample sufficient for theanalysis, the rest of the liquid required for the injection being aliquid available in the analysis apparatus, and not as precious as thesample.

The sleeving is particularly useful when using a tank with a widesection while maintaining a small section for the sample flow. As one ofthe means for adjusting the sample flow in relation to the sleevingflow, the device can advantageously comprise means for adjusting a flowrate of the displacement liquid with respect to the flow rate ofsleeving liquid. The adjustment means can comprise means for a pressuredrop in a branch circuit for the displacement liquid and/or means for apressure drop in a branch circuit for the sleeving liquid. For example,the pressure drop means can be chosen from a known length of acalibrated tube, a fixed hydraulic resistance and a variable resistance.

The hydraulic device can comprise only one motorization, for example asingle electric motor, in order to generate the sample flow and thesleeving flow simultaneously. Moreover, it can comprise at least twosyringes in order to generate the sample flow and the sleeving flow, thesyringe pistons being firmly attached to each other. They thus have acommon movement and the sample and sleeving flows are indeedsimultaneous.

In particular, a hydrofocus tank from the prior art can be used with acircuit such as described previously according to the invention, theinjection of the sample into this tank can take place without pressurerelative to the sleeving flow.

According to the invention, a method for the analysis of a blood samplein a flow-through cytometer is also proposed, characterized in that ablood sample is injected, optionally under pressure, into a flow-throughtank of the cytometer, the sample forming a sample flow there and aliquid sleeving flow is created around the sample flow, with a sleevingliquid, characterized in that the flow rate of the sample flow isadjusted with respect to the flow rate of the sleeving liquid.

In particular, it is possible to introduce the sample into an injectionbranch of a hydraulic circuit, and to introduce upstream of the samplein the injection branch, a displacement liquid, the displacement liquidserving to push the sample during its injection into the tank. Thisdisplacement liquid can be chosen from a reagent and a diluent,preferably a reagent. There is therefore no point in providing a liquidother than that which is strictly necessary for the preparation of thesample with a view to its analysis or analyses.

It is also possible to create around the sample flow in the tank, asleeving flow with a sleeving liquid. This sleeving liquid can also bechosen from a reagent and a diluent, preferably a diluent. In this casealso, there is not point in providing a liquid other than those whichare strictly necessary for the preparation of the sample with a view toits analysis or analyses.

In the case where a hydrofocus method, or a tank according to the thirdobject of the invention, is used, it is advantageous to adjust the flowrate of displacement liquid with respect to the flow rate of thesleeving liquid, for example by introducing a pressure drop in a branchcircuit for a displacement liquid and/or pressure drop means in a branchcircuit for a sleeving liquid.

In a method according to the invention, in particular for a tankaccording to the third object of the invention, it can easily beprovided that the blood sample has a dilution rate of at least1/100^(th). In fact, in such a method, the sample can be introducedunder pressure relative to the sleeving liquid, into the tank, at avelocity greater than that of the methods of the prior art, and withgreater section widths for the sample flow in the tank. Thus, withoutincreasing the analysis time, for a differentiation and a counting ofthe leucocytes, a dilution rate can be used which is identical to thatused conventionally for the measurement of haemoglobin, in particulardilution rates comprised between 1/100^(th) and 1/500^(th), particularlybetween 1/160^(th) and 1/180^(th).

The invention also relates to a haematology apparatus, in particular anautomatic blood analysis apparatus, characterized in that it comprises ahydraulic device according to the invention.

The present invention will be better understood and other advantageswill become apparent in light of the following description ofembodiments, which description is made in particular with reference tothe attached drawings in which:

FIG. 1 diagrammatically illustrates an example of equipment according tothe first object of the invention;

FIGS. 2 a-2 e are graphs of linearity tests of the measurement ofhaemoglobin by spectrophotometry according to the method of theinvention;

FIGS. 2 f-2 i are corresponding cytographs;

FIG. 3 is a diagrammatic view of an automatic apparatus for analysis ofa blood sample using a hydraulic device according to the fourth objectof the present invention;

FIG. 4 is a diagrammatic longitudinal view of an optical device unitaccording to the second object of the invention;

FIG. 5 is a more detailed diagrammatic longitudinal view of the opticaldevice of FIG. 4, in a plane perpendicular to that of FIG. 4;

FIG. 6 is a perspective view of an optical tank according to the thirdobject of the invention;

FIG. 7 is a longitudinal section view of a first embodiment of aninjector for an optical tank according to the invention;

FIG. 8 is a longitudinal section view of a second embodiment of aninjector for an optical tank according to the invention;

FIG. 9 is a longitudinal section view of one end of the injector of FIG.8;

FIG. 10 is a longitudinal section view of a tank illustrating a methodof the prior art for injecting the blood sample into the tank; and

FIGS. 11 a-11 c are graphs illustrating results obtained with anautomatic apparatus using the method of the invention and using acytograph with the optical device and tank according to the invention.

FIG. 1 diagrammatically illustrates a single dilution and analysis tank1 which can be supplied with a blood sample 2 to be analyzed, a diluent3 and a reagent 4 together forming an analysis solution. This tank 1 isequipped with means for measuring by photometry 5 the haemoglobin levelin said analysis solution and means for measuring 6 the resistivity ofsaid analysis solution in order to count the total number of leucocytes.Means are generally provided for taking a fraction of the analysissolution from the analysis tank 1 and for injecting it into an opticaltank 7 equipped with optical measurement means 8 (for example a flowcytometer) for an analysis of the leucocytes. According to the examplechosen, means are also provided for taking a fraction of a pre-solutionconstituted by the sample of blood and diluent, and introducing it intoa counting and dilution tank 9 equipped with means for measuring theresistivity 10 of said fraction in order to count the erythrocytes andplatelets. The equipment is conventionally equipped with heating meansin order to obtain a thermostatically-controlled temperature ofapproximately 35° C. This temperature allows optimal lysis reaction timeand quality of the erythrocytes.

The equipment-operates in the following manner:

one aliquot of blood (15.6 μl) is injected into the analysis tank 1 anddiluted with 2 ml of diluent so as to form an analysis pre-solution; thedilution rate is 1/130^(th);

a very small fraction (approximately 20 μl) is taken from this analysispre-solution and deposited in the tank 9 for counting the erythrocytesand platelets;

0.7 ml of reagent is then added to the remaining pre-solution in theanalysis tank 1: the lysis lasts for approximately 10 seconds (in orderto destroy the erythrocytes, form and stabilize the oxyhemoglobincomplex), the analysis solution thus formed has a final dilution rate ofapproximately 1/173^(rd); a fraction of said analysis solution is takenand injected into the optical tank 7 where the analysis of theleucocytes can take place (counting and/or differentiation of theleucocytes by sub-populations); simultaneously in the analysis tank 1,the leucocytes are counted by a resistivity measurement and thehaemoglobin by a measurement by absorbance at the wave length of theoxyhemoglobin complex formed.

An optical device according to the invention, particularly suitable fora leucocytic analysis of an analysis solution having a dilution ratelower than 1/100^(th) is described below, more particularly suitable fora dilution comprised between 1/160^(th) and 1/180^(th). Conventionally,a dilution rate of 1/160^(th) is considered to be lower than a rate of1/100^(th).

Of course variant embodiments of the method and of the equipmentdescribed above are possible:

for the equipment: means can be provided for separately introducing thelysis compound, the leucoprotective compound and the compoundstabilizing the complex formed with the haemoglobin in the analysis tank1, and therefore rather more in the form of a mono-reagent; the means 6for measuring the resistivity of the analysis solution are optional; thetotal number of leucocytes being able to be obtained by optical analysisof the analysis solution; similarly the counting tank 9 and the meansfor measuring 10 the resistivity in this tank can be provided only if acomplete analysis of the blood sample is desired;

likewise for the method: the introduction of the reaction compounds canbe envisaged independently or collectively in place of a mono-reagent,the introduction being able to be carried out simultaneously orsuccessively; the previous stage of counting the erythrocytes andplatelets and the stage of global counting of the leucocytes can beomitted; moreover, two successive dilutions of the blood sample can becarried out: a first dilution which is particularly suitable for aleucocyte differentiation (approximately to 1/80^(th)) as takes place inthe known standard hydrofocus-type cytometer, from which the fractionrequired for this leucocyte differentiation is taken, then at a secondmoment in time a second dilution suitable for measurement of thehaemoglobin (comprised between 1/100^(th) and 1/500^(th)) as is possiblewith the known spectrometers.

According to yet another variant, the tank 1 can serve at a secondmoment in time to carrying out counting the erythrocytes and plateletsafter cleaning, by filling the tank with a sample waiting in a syringeneedle.

The results obtained will now be described with a specific example of(mono)-reagent according to the invention:

A mono-reagent is prepared using the Eosinofix® formulation from thecompany ABX marketed for leucocyte determination in flow cytometry andcontaining for this purpose a compound for lyzing the erythrocytes and aleucoprotective compound (cf. patent EP0430750 by ABX). According to theinvention, a compound stabilizing the haemoglobin complex was added.

Measurement of the Haemoglobin by Spectrophotometry:

Linearity tests were carried out using a spectrophotometer at 542 nm.The graphs are shown in FIGS. 2 a-2 e. They represent the haemoglobinconcentrations measured in relation to the expected concentrations. Morespecifically:

-   -   FIG. 2 a corresponds to a reference lysis for measurement of the        haemoglobin by spectrophotometry (LMG® sold by the company        ORPHEE);    -   FIGS. 2 b, 2 c and 2 d correspond to the mono-reagent according        to embodiment No. 4 with, as stabilization agent of the        haemoglobin complex, respectively Tiron, DDAPS and imidazole;        and    -   FIG. 2 e corresponds to the method of the invention implemented        using as mono-reagent Eosinofix® alone, i.e. containing no        stabilization agent of the haemoglobin complex according to the        present invention.

For the three tests carried out according to the invention, a positivelinearity test is obtained for each with a correlation coefficient R² of1±10⁻⁴ (shown in the figure). This result is in accordance with thatobtained with the reference lysis of FIG. 2 a. This means that themethod of the invention does indeed allow measurement of a realhaemoglobin level in a blood sample.

By contrast, as is seen in FIG. 2 e, with the reagent (Eosinofix)without a hemoglobin stabilizer, a linear relationship is not obtained.This means that this reagent alone cannot be used to measure ahaemoglobin level.

Leucocyte Differentiation by Flow Cytometry

FIGS. 2 f to 2 i are cytographs obtained using a BD FACScan® flowcytometer, corresponding respectively to Eosinofix alone and Eosinofixto which DDAPS, Tiron and imidazole are added. In these figures, it isseen that the differentiation of the sub-populations is indeed achievedand in a manner which is comparable to a standard reagent for leucocytedifferentiation (matrix obtained with Eosinofix in FIG. 2 f).

Reference can also be made to the cytograph of FIG. 11 b (describedbelow) in particular obtained with a cytometer according to theinvention.

The hydraulic device according to the fourth object of the inventionwill now be described.

FIG. 3 partially represents the diagram of a hydraulic system 100 andsome of the equipment of an automatic blood analyzer 20, in so far as itallows an understanding of the hydraulic device according to theinvention.

The automatic apparatus illustrated in FIG. 3 in particular comprises aneedle 101 for sampling blood to be analyzed in a tube which was usedfor its storage and its transport to the automatic apparatus. The bloodtaken is poured by the needle in the form of a sample into a tank 102.The tank 102 is in particular designed for the dilution and/or the lysisof the erythrocytes of the blood sample. All or part of the sample,before or after dilution, can be taken with a view to analysis inanother part of the automatic apparatus, for example in a device 120,described below. A device for analysis of the haemoglobin 110 (aspectrophotometer for example) is arranged close to the tank 102. Astore 103 for a dilution product and a store 104 for a reagent, inparticular a lysis reagent are connected to the tank 102 via thehydraulic circuit 100.

Another analysis device 120 is more specifically dedicated to thecounting and differentiation of the leucocytes, for example on the wholeor part of the sample taken from the tank 102. Hereafter sample willalso refer to this whole or this part. The device for analysis of theleucocytes 120 in particular comprises an optical device 200 and anoptical tank 300. The optical tank is connected to the tank 102 via thehydraulic circuit.

A set of syringes allows the movement of the liquids in the hydrauliccircuit. Of these syringes, a syringe 105 dedicated to the diluent and asyringe 106 dedicated to the reagent are represented so that theinvention is well understood. Other syringes which are not representedbecause they are not necessary in order to understand the invention cancomplete the device.

Besides the pipes for the circulation of the liquids, the hydrauliccircuit comprises solenoid valves for the change-over of differentcircuits in the hydraulic circuit 100, according to its use at a givenmoment of the analysis. Eight solenoid valves 111-119 of the solenoidvalves of the hydraulic circuit 100 are illustrated in FIG. 3. Eachsolenoid valve comprises two positions, each labelled respectively withthe letter A or B.

The design of the hydraulic circuit as will be described below, allowsthe use of only one motorization M for the syringes illustrated. Thesame motorization can also be used for other syringes. Thus, the pistonsof the syringes 105, 106 are firmly attached to each other. Theirmovement is therefore simultaneous, either pushing P, when they aredriven into the respective cylinder of each syringe, or pulling T whenthey are withdrawn.

The arrangement and then the hydraulic operation of the automaticapparatus will now be described.

The tank 300 comprises an external body 301 and an injector 302, insidethe body 301, a sleeving volume 303 is formed between the body and theinjector.

The hydraulic circuit 100 comprises:

an injection branch 131 which extends upstream of the injector, betweenthe injector and the valve 111;

a sample branch 132 which is connected at a sample branching point 142to the injection branch and extends to the tank 102;

a suction branch 133 which is connected at a suction branching point 143to the injection branch, upstream of the sample branching point 142, viathe valve 113 and extends to a vacuum source 107, for example a syringeor a peristaltic pump;

a discharge branch 134 which is connected at a discharge branching point144 to the injection branch, upstream of the suction branching point143, and extends to the reagent product store 104;

a sleeving branch 135 which extends upstream of the body 301 andconnects the sleeving volume and the valve 115;

a dilution branch 136 which extends between the valve 116 and a use 108for the diluent via the valve 115;

a diluent branch 137 which extends between the diluent store 103 and thevalve 116;

a reagent branch 140 which extends between the reagent store 104 and thevalve 117;

a reaction branch 141 which extends between the valve 117 and a use 109for the reagent via the valve 111;

a draining branch 138 for the tank 102 which extends between the tank102 and the vacuum source 107 via the valve 118, the sample branch 132being connected with the draining branch between the tank 102 and thevalve 118, and the suction branch being connected to the outlet branch132 beyond the valve 118 in relation to the tank; and,

an outlet branch 139 which connects the downstream of the tank 300, viathe valve 119, to a waste tank, for example at atmospheric pressure orvia a suction source, a syringe or a peristaltic pump.

In a first position 116A of the valve 116, the dilution syringe 105 isin communication with the diluent store, so that a pulling movement Tallows the syringe 105 to be filled with diluent.

In a first case the dilution syringe containing diluent, with the valve116 being in its second position 116B which connects the syringe 105 tothe dilution branch 136 and the valve 115 being in its first position115A which connects the dilution branch to the use 108 for the diluent,a pushing movement P allows the diluent to be moved to this use 108, forexample in the tank 102, for example for a dilution of the whole sample.

In a second case, with the valve 116 being in its second position 116Band the valve 115 being in its second position 115B which connects thedilution branch to the sleeving branch 135, a pushing movement P allowsthe diluent to be moved into the optical tank 300, in order to form asleeving flow there. The usefulness of this sleeving flow in the contextof the invention will be analyzed in a description of the tank 300below.

The valve 117 being in a first position 117A which connects the syringeof reagent to the reagent store 104, and the valve 114 being in a firstposition 114A which shuts off the discharge branch 134, a pullingmovement T allows the reagent syringe 106 to be filled with reagent.

In a first case, the reagent syringe containing reagent, with the valve117 being in its second position 117B which connects the reagent syringe104 to the reaction branch 141 and the valve 111 being in a firstposition 111A which connects the reaction branch to the use 109 for thereagent, a pushing movement P allows the reagent to be moved to this use109, for example in the tank 102, for example for a lysis of the wholesample.

In a second case, the valve 117 being in its second position 117B andthe valve 111 being in its second position 111B which connects thereaction branch 141 to the injection branch 131, the reagent syringe 106is directly connected to the injector 302.

The valve 118 being in a first position 118A which isolates the suctionbranch 133 from the sample branch 132 through the draining branch, thevalve 112 being in a first position 112A which connects the upstreampart to the downstream part of the sample branch 132, the valve 113being in a first position 113A which connects the downstream part to theupstream part of the suction branch 133, therefore to the vacuum source107, the sample to be analyzed is sucked into the injection branch 131,between the sample branching point 142 and the suction branching point143.

The discharge branch 134 comprises a variable or calibrated fluidresistance 150.

When the diluent syringe 105 contains diluent, the reagent syringe 106contains reagent and a blood sample to be analyzed is in the injectionbranch 131; and when furthermore the valves 112, 113 are in their secondpositions 112B, 113B which isolate the upstream part and the downstreampart from their respective arms; and when the valves 115, 116 are intheir second positions 115B, 116B which connect the diluent syringe 105to the sleeving volume 303; when finally the valves 111, 117 are intheir second positions 111B, 117B which connect the reagent syringe 106to the injector 302 and the valve 114 is in its second position 114B; asingle pushing movement P generated by the single motorization M, allowsthe driving of the diluent, the reagent and the blood sample in thedirection of and through the tank 300, while a part of the reagent,which is a function of the fluid resistance 150, is returned to thereagent store 104.

The resistance 150 in particular allows adjust of the flow rates of thesleeving and displacement liquids one with respect of the other. Thisallows these flow rates to be adapted to the different functions ofthese liquids. In particular, this allows similar flow velocities to beobtained for the sleeving and the sample in the analysis zone 304 when astandard hydrofocus tank is used.

In particular, the discharge branch 134 and the arrangements describedpreviously make it possible to use a single motorization and thereforeto reduce in particular the cost of an automatic analysis apparatus, aswell as its bulk.

The diluent forms, in an analysis zone 304 of the tank 300 a sleevingflow for the sample (see in particular FIGS. 4 and 5). The reagent,situated upstream of the sample in the injection branch 131, serves as adisplacement liquid, i.e. it allows the piston movement of the reagentsyringe to be transmitted to the sample. Thus, there is no point infilling the reagent syringe with the sample in order to be able toundertake its analysis. Thus, even a sample of small volume can beanalyzed, and all of this sample can be injected and analyzed withoutsome of it remaining in the injection branch 131 or in the syringe 106.

Of course, other syringes, valves and branches, not represented in FIG.3, can make up the hydraulic circuit 100, in order for the automaticanalysis apparatus 20 to operate fully and well.

The optical device 200 according to the invention will now be described,in particular with regard to FIGS. 4 and 5.

The optical device comprises an approximately monochromatic light source201. This light source is an electroluminescent diode. The light isprincipally emitted along an optical axis X200. The optical axis X200 isarranged approximately perpendicular to an injection axis X300 formovement of the sample in the optical tank 300. The two axes X200 andX300 together define an optical plane.

In order to prevent the source light beam 311 produced by the source 201from being polluted by spurious light, a set of three diaphragms, isarranged, each one perpendicular, on the path of the beam. Thediaphragms 202 are pierced with holes the diameter of which isapproximately equal to the beam and is progressively increased in eachdiaphragm in order to adapt it to the diameter of the measurement beamas this diameter increases the further away from the source 201 it is.The beam then passes through a focusing device 203 constituted by one ormore lenses.

Beyond the focusing device the beam encounters an adjustment devicewhich allows the optical axis to be moved in a plane perpendicular tothe injection axis X300, i.e. in a transverse direction in relation tothe movement of the sample in the tank. A lateral shift of the beam canlead to a partial or no illumination of the sample which has a directinfluence on the analysis result.

In the context of the example described, the adjustment device isconstituted by a transparent slide 220 rotatably mounted about an axisX220. The axis 221 is approximately parallel to the injection axis X300.If the slide is arranged perpendicular to the optical axis X200, thebeam passes through it without being deflected. By contrast, if theslide forms an angle with the optical axis, a double refraction, atentry and exit of the slide, shifts the beam in a plane perpendicular tothe adjustment axis X220. The adjustment axis X220 being approximatelyparallel to the injection axis X300, only a transverse shift isgenerated by the refraction in the slide. The greater the thicknessand/or the refractive index of the slide and the more the slide isinclined with respect to the optical axis the greater is the shift.Thus, for a slide with a chosen thickness and refractive index, it issufficient to rotate the slide 220 about its axis X220 in order toadjust the position of the beam relative to the sample which moves inthe analysis zone 304 of the optical tank 300. Such an adjustment deviceis particularly economical compared to the devices of the prior art,especially as a precise rotation is generally easier to carry out than aprecise translation, using high-precision mechanics.

After having penetrated the tank and passed through the sample, thesource beam 211 at least partially becomes an axially-resulting beam212, which exits the tank approximately along the optical axis. Theaxially-resulting beam 212 carries information about the sample which ithas passed through.

In order to allow simultaneous measurements of several of these items ofinformation it must be possible to analyze the beam with severalmeasurement apparatuses 222, 223. In particular, the optical analysisrelies on the detection of the light diffracted according to two rangesof angles: narrow angles and wide angles. In each of the ranges ofangles, two different items of information are used. It is thereforenecessary to distribute the light in two different channels for eachrange. Therefore means 205 for separating the resulting beam 212 intotwo resulting beams 213, 214 are used. The separation means are mainlyconstituted by a beam splitter 205. This beam splitter is a transparentglass slide. It is arranged at 45 degrees to the optical axis. Asecondary axially-resultant beam 213, formed by the light which haspassed through the beam splitter, and a beam resulting from loss 214formed by the Fresnel losses, i.e. by the light reflected by the beamsplitter, are thus produced.

Such a beam splitter has a very low cost compared to the separationmeans used in the prior art in optical analysis devices of this type. Inparticular, because it does not comprise any additional reflectivecoating, it is virtually age resistant and requires practically nomaintenance. Given the multiple reflections inside the slide and thepolarization of the incident radiation of the axially-resulting beam,between 5 and 15% of the energy is reflected, the rest being transmittedin the form of the secondary axially-resulting beam.

Between the tank and the beam splitter, the axially-resulting beam 212is rendered parallel by suitable means 206. Beyond the beam splitter,the resulting beams 213, 214 are again focussed by respective suitablemeans 207, 208, with a view to their analysis by the respectivemeasurement apparatuses 222, 223.

In the example described, the measurement apparatus 222, which analyzesthe secondary axially-resulting beam 213 is an apparatus for measurementof the diffraction close to the optical axis by the blood cells (calledan FSC measurement). In the example described, the measurement apparatus223, which analyzes the beam produced by the Fresnel losses 214 is anapparatus for measurement of the light losses in the axis (called an ALLmeasurement), i.e. the obscuring of the light by the cells in thesample.

FIG. 5 diagrammatically represents a section of the tank in a planeperpendicular to the injection axis X300 and containing the optical axisX200. As is particularly illustrated in this figure, the light reemittedlaterally by the sample in a laterally-resulting flow 315, focussedbeyond the tank in a measurement apparatus 224, is also analyzed.

An optical tank according to the invention, in particular envisaged foruse with a hydraulic circuit such as described previously, will now bedescribed, in particular with reference to FIG. 6. The operation of thistank can be compared with a hydrofocus-type operation of the prior art,which is represented very diagrammatically in FIG. 10.

The tank 350 of FIG. 10 comprises a body 351, an injector 302 and ananalysis zone 354. An internal transverse dimension D354 of the tank isapproximately 250 microns. This dimension can be a diameter, if the tankhas a circular section, or one side, if it has a square or rectangularsection. As illustrated by the dotted lines, a sleeving flow 362 is usedto reduce in particular the diameter of a sample flow 361, so that inthe analysis zone 354 the sample flow has, in the prior art, a diameterD361 of less than 50 microns.

The tank 300 according to the invention, illustrated in FIGS. 4-6,comprises the body 301 and the injector 302, arranged approximatelycoaxially along an injection axis X300. The analysis zone 304 isarranged downstream of the injector.

The body is produced from an injected material, preferably from aplastic material. Such a production method allows complex shapes to beobtained. In particular, a lens 305 is moulded in the body. This lensallows the light which is obscured, diffracted or diffused by the bloodcells to be collected.

This lens must have dimensions, in particular sufficient diameter forthe possible local inhomogeneities in the injected material to benegligible in relation to these dimensions. In the example illustrated,the lens 305 has a diameter of about 3 mm. This injected lens is alateral lens 305 which the laterally-resulting beam 315 passes through.Moreover, the lateral lens must allow the light to be collected in asmany directions as possible, i.e. with a directional field which is aslarge as possible. Thus, the closer the lens is to the sample, thegreater the directional field. In the example illustrated, the lens is ahemispherical lens, called a 90° lens. Moreover, the lens being a partof the wall of the tank, there is direct contact with the liquid in thetank, i.e. there is no air space, with a low refractive index, betweenthe sample and the lens. This improves the measurement.

In order to overcome the homogeneity deficiencies, glass is used wherethe light is particularly focussed, for example a glass of the BK7 type.This is the case in particular for the axial windows 306, where thesource beam 211 penetrates the tank and where the axially-resulting beam212 exits it.

In order to be able to produce an injected lens with such dimensions, itis necessary, in the analysis zone, for the tank 300 to have at leastcomparable dimensions. Moreover these large dimensions allow glasswindows to be integrated into plastic walls, while the tanks of theprior art, having small dimensions, are made with walls entirely ofglass or quartz. In the example illustrated in particular in FIGS. 5 and6, the lower section of the tank is 4.5 mm along the optical-axis by 3mm in the perpendicular-direction. This rectangular section with largedimensions associated with a small volume of the sample, whichtransports the blood cells to be analyzed, requires the use of ahydrodynamic sleeving of the sample. By way of comparison, a tank of theprior art has an internal transverse dimension D354 of the analysis zoneclose to 250 microns.

Upstream of the analysis zone 304, the body 301 of the tank surroundsthe injector 302 and forms around the injector the sleeving volume 303.The walls of the injector separate a flow 311 formed by the sample,inside the injector, from a sleeving flow 312, in the sleeving volume.The sample flow originates from the injection branch 131 of thehydraulic circuit 100. The sleeving flow originates from the sleevingbranch 135 of the hydraulic circuit. In the analysis zone, the two flowsare in contact, remain concentric and flow simultaneously in the tank.

In order to reduce the production costs of the automatic apparatus, itcan be advantageous to reduce the precision of the production of theparts. As mentioned above, such an aim can be achieved by creating asample flow with a larger section.

However, if a technique of the prior art is used where the sample flowis stretched by a sleeving flow, a sample flow with a large section willbe turbulent, which in particular adversely effects the precision ofmeasurements. Moreover the section of the sample flow will beprogressively reduced, which is the opposite of the effect desired,which is to have a sample flow with a large section. Such an aim isachieved by using the hydraulic circuit 100 according to the invention,described previously with reference to FIG. 1. Such a circuit makes itpossible to obtain independently chosen velocities for the flow of thesleeving flow and for that of the sample flow, in order that littleturbulence appears in the sample flow and that this turbulence has nonotable effect on the analysis result. The two flows can each beapproximately uniform, optionally laminar in certain appropriatevelocity ranges.

Moreover, an injector 302 as illustrated in FIG. 7 or FIG. 8 also allowslimitation of the turbulence in the sample flow. Moreover, it allows ahigh velocity of injection of the sample into the optical tank, whileretaining its flow approximately uniform.

An injector 302 as illustrated in FIG. 7 comprises a structural tube320, for example made of stainless steel ensuring the stiffness of theinjector. The structural tube is sheathed on the inside with a tube 321made of a plastic, for example a polytetrafluoroethylene (PTFE). In theexample illustrated the structural and sheathing tubes are cylindrical.The sheathing tube is extended, downstream of the structural tube, by anozzle made of the same plastic material. The fact of differentiatingthe structural function of the structural tube and the injectionfunction of the nozzle, associated with the use of a plastic material,allows sufficiently precise shapes to be obtained at a low cost.

The nozzle has a section which is progressively narrowed from aninternal diameter D321 of the sheathing tube to an internal diameterD323 of an outlet orifice 323 at a downstream end 324 of the nozzle 322.In the example described, the downstream end 324 is a cylinder with alength L324. The wall of the nozzle is initially inwardly concave, theninflected to become inwardly convex, the section of the nozzle thusbeing progressively narrowed from the upstream to the downstream, fromthe diameter D321 to the diameter D324. The concave surface is tangentto the inner surface of the cylindrical sheathing tube. The convexsurface is tangent to the inner surface of the cylindrical end 324. Inthe example described, the diameter D323 of the orifice 323 isapproximately 60 microns, the internal diameter D321 of the sheathingtube is approximately 1 millimetre, the length L322 of the nozzle isapproximately 2.5 millimetres, that L320 of the structural tube isapproximately 6 millimetres and that of the cylindrical end L324approximately 200 microns.

An injector 302 such as that illustrated in FIGS. 8 and 9 is in a singlepiece and made of a single substantially stiff material. This materialcan be, for example, stainless steel, a ceramic, a synthetic ruby or aplastic material. The plastic material can advantageously be apolytetrafluoroethylene. The injector comprises an approximatelycylindrical tube 331 which is extended downstream by a nozzle 332.

The nozzle progressively narrows inwardly, from an internal diameterD331 for the tube 331, to an internal diameter D333 of an outlet orifice333 for the sample, at a downstream end 334 of the nozzle 332. In theexample illustrated, the narrowing takes place according to a truncatedcone open at an angle preferably comprised between 9 and 10 degrees.Beyond the truncated cone and up to the outlet orifice 333, the diameterremains constant in a cylindrical part 335, with a length L335 and adiameter D333.

On the exterior of the nozzle, its external diameter is progressivelylarger according to a truncated cone open at an angle comprised betweenapproximately 8 and 9 degrees, then, in the noticeably more reduced endaccording to a truncated cone open at an angle A334 comprised betweenapproximately 35 and 45 degrees, to an external diameter D334 around theoutlet orifice 333. D334 is approximately 3 to 4 times larger than D333.

By way of example D333=60 μm, D334=200 μm and A334=40°.

Thanks to the different arrangements described previously, it ispossible to obtain a high injection velocity. Thus, in the exampledescribed it is possible to inject a sample of more than 200 microlitresin less than 10 seconds. In particular, such an injection rate makes itpossible to use a high rate of dilution of the blood sample, withoutincreasing the duration of the analysis compared to automaticapparatuses of the prior art. In particular, the same dilution, forexample 1/160^(th), can be used for the analysis of the haemoglobin bythe device 110 (see FIG. 3), and for the analysis of the leucocytes bythe optical device 120, instead of 1/80^(th) generally used for theanalysis of the leucocytes.

FIGS. 11 a-c illustrate the results obtained using the method and theequipment according to the first object of the invention, said equipmentusing an optical tank 7 according to the third object of the inventionand an optical device 8 according to the second object of the invention.FIG. 11 a shows a positive linearity test of the haemoglobin measurementand therefore demonstrates the possible and reliable measurement of thehaemoglobin level of a blood sample according to the invention. FIG. 11b shows an optical matrix obtained from a test sample of blood with 30%eosinophils to which the formulation according to the invention has beenadded. On this matrix, the five sub-populations are present anddifferentiated (groups delimited in the cytograph: E for eosinophils, Nfor neutrophils, M for monocytes, B for basophils and L forlymphocytes). FIG. 11 c shows the positive linearity test of themeasurement by resistivity of the level of leucocytes.

These figures show that thanks to the invention it is possible to carryout an analysis of at least the level of haemoglobin and the level ofleucocytes and a leucocyte differentiation using the formulationaccording to the invention, in particular in the form of a mono-reagent.

Of course, the invention is not limited to the examples which have justbeen described and numerous modifications can be applied to theseexamples without exceeding the scope of the invention.

For example, products other than the diluent or the reagent can be usedin order to form respectively the sleeving flow and the fluid piston,particularly if they are available in the automatic apparatus for otheruses.

In addition, instead of being arranged only on the injection circuit, afluid resistance can be arranged on the sleeving circuit or on both ofthese simultaneously. This can occur as a function of the given maximumflow rate via the means for displacement of the liquids intendedrespectively for displacement or sleeving.

Several or all of the lenses of the optical tank and/or of the opticaldevice can thus be produced by injection with the body of the tank,instead of a single one as illustrated previously. In particular, theglass windows can be injected. Particularly if the inhomogeneities inthe injected material are more or less negligible with regard to theprecision desired for the measurements.

An adjustment device and/or the separation means described previouslycan be used independently of each other and optionally with a lightsource other than an electroluminescent diode.

1. Method for the automatic analysis of a blood sample in which: ananalysis solution containing said blood sample, a diluent, and: at leastone compound to lyze the erythrocytes; at least one compound tostabilize the haemoglobin in the form of a chromogenic complex, isformed in a single dilution and analysis tank, the haemoglobin level ismeasured in this analysis solution by spectrophotometry in said tankafter the lysis of the erythrocytes; and an appropriate quantity of thisanalysis solution is taken from said tank on which a leucocyticdifferentiation is carried out by an optical means, characterized inthat the analysis solution also contains at least one compound toprotest the leucocytes, allowing the distinguishing of at least fourmain leucocyte sub-populations
 2. Method according to claim 1,characterized in that the analysis solution contains at least onecompound to stabilize the haemoglobin in the form of oxyhaemoglobin. 3.Method according to claim 1, characterized in that a dye or mixture ofdyes specifically labelling at least one leucocyte sub-population isalso added to the analysis solution.
 4. Method according to claim 1,characterized in that as said compounds are chemically compatible, theyare added in order to form the analysis solution in the form of amono-reagent.
 5. Method according to claim 1, characterized in that themono-reagent is suitable to carry out the function of diluent in orderto produce a mono-dilution of the blood sample.
 6. Method according toclaim 1, characterized in that the analysis solution has a dilution rateof the blood sample suitable for measuring the haemoglobin byspectrophotometry.
 7. Method according to claim 6, characterized in thatthe dilution rate of the analysis solution is comprised between1/100^(th) and 1/500^(th), and preferably between 1/160^(th) and1/180^(th).
 8. Method according to claim 1, characterized in that thenumber of leucocytes in said measuring solution in said tank is countedby resistivity.
 9. Method according to claim 1, characterized in thatthe number of leucocytes in said measuring solution in said tank iscounted using the optical means.
 10. Method according to claim 1,characterized in that the sampling of an appropriate quantity ofanalysis solution to carry out the leucocyte differentiation is carriedout before the measurement of the haemoglobin in said tank, the dilutionrate of the sampled measuring solution being suitable for saidmeasurement by said optical means, and in that diluent is added to saidremaining analysis solution so as to increase the dilution in said tankin order to obtain a dilution suitable for a measurement of thehaemoglobin by spectrophotometry.
 11. Method according to claim 10,characterized in that the optical means is a hydrofocus-type flowcytometer, and the dilution rate of the sampled analysis solution formeasurement by cytometry is less than 1/100^(th).
 12. Method accordingto claim 10, characterized in that the optical means is a cytometer withpassive sleeving, and the dilution rate of the sampled solution formeasurement by cytometry is greater than 1/100^(th).
 13. Methodaccording to claim 10, characterized in that the dilution rate of themeasuring solution for the measurement of the haemoglobin byspectrophotometry is comprised between 1/100^(th) and 1/500^(th). 14.Method according to claim 1, characterized in that in a stage before theaddition of said compounds, a fraction of a pre-solution is takenconstituted by the sample of blood and diluent on which counting of theerythrocytes and/or platelets is carried out by resistivity.
 15. Methodaccording to claim 1, characterized in that lysis compound is an ionicsurfactant chosen from: the quaternary ammonium salts, preferablyalkyltrimethylammonium salts and still more particularly cetyl-,dodecyl-, tetradecyl- and hexadecyltrimethylammonium bromides andchlorides; pyridinium salts; long-chain ethoxylated amines; and alkylsulphates (SDS).
 16. Method according to claim 1, characterized in thatthe compound which stabilizes the haemoglobin in the form of achromogenic complex is preferably chosen from: mono or polydentatechelates presenting ligand atoms (non-binding pairs: O, N, S, andcarboxy COO— groups etc.), the aromatic compounds (mono or polydentatechelates) comprising ligand atoms (having non-binding pairs: O, N, Setc.), and saponins, tertiary amine oxides, betaines and sulphobetainesof quaternary ammoniums.
 17. Method according to claim 1, characterizedin that the leucoprotective compound is a non-ionic or amphotericsurfactant preferably chosen from: the ethoxylated alcohols, betainesand sulphobetaines of quaternary ammoniums, tertiary amine oxides, theglycosidic type compounds and the glucidic type compounds.
 18. Methodaccording to claim 1, characterized in that a fixing agent of themembrane of the leucocytes is also added to the analysis solution. 19.Method according to claim 1, characterized in that a pH buffer capableof setting the pH of the analysis solution between 5.0 and 8.0,preferably at a pH of 7, is also added to the analysis solution. 20.Method according to claim 1, characterized in that a background salt isalso added to the analysis solution to ensure its conductivity. 21.Haematological analysis apparatus for the implementation of the methodaccording to claim 1, characterized by: a single dilution and analysistank (1) suitable for receiving said analysis solution; a means formeasurement (5) by spectrophotometry in said tank of the haemoglobinlevel in said analysis solution; a means of sampling said analysissolution; and a means for the optical measurement (7, 8) of said samplein order to carry out a leucocyte analysis.
 22. Apparatus according toclaim 21, characterized in that it comprises means for counting theleucocytes (6) by resistivity in said tank (1).
 23. Apparatus accordingto claim 21, characterized in that it comprises means for carrying outthe counting of erythrocytes and platelets (9, 10).
 24. Apparatusaccording to claim 21, characterized in that it comprises a countingtank (9) for the erythrocytes and platelets and means for measuring (10)the resistivity in said counting tank.